We continued some of the projects that were started during the previous year but also initiated new collaborations with several intramural researchers. We continued our collaboration with NCI on the structure/stoichiometry of the centromere. There is an ongoing controversy as to the stoichiometry of histones in the centromere. Based on biochemistry, AFM and EM imaging it has been proposed that the centromere contains just a tetramer of histones as opposed to the standard octameric nucleosome. Our work established that actually the centromere is a dynamic structure during the cells cycle alternating between an octamer and a tetramer before and after DNA replication. One of the arguments supporting this work has been the height of the nucleosomal particles as measured by the AFM. There was a recent publication claiming that the change of heights is indeed there but that it is due to other factors, such as conformational changes in the histones. We and another laboratory outside NIH went back and examined those results and found inconsistencies but without arriving at a definitive conclusion as to the reasons behind the height changes. Hence the controversy continues. A letter manuscript describing this work was accepted by Nature, Structural Molecular Biology to stimulate further discussion on the issue. The work continues in the direction of developing a protocol for co-localized optical and AFM imaging of nucleosomes using quantum dots as the histone H3 beacons. Our collaboration with the NICHD on the mapping of composition and mechanics of cartilage continued and we have completed the data acquisition and analysis for the elasticity variation in the axial direction. The obtained axial elasticity profiles correlate with composition variations and we are preparing a manuscript on the basic methodologies developed for the study of cartilage. At this point we are starting to examine age related changes as well as osteoarthritic tissue aiming at better understanding of function and of disease. In addition we hope to advance new hypotheses on the mechanisms of tissue degradation with age and in disease. We continued to investigate the role of various bacterial proteins (such as GalR, CI and HU) in packaging bacterial DNA and in controlling transcription. We imaged a number of protein/DNA complexes and used image analysis to establish looping driven by specific protein binding and/or multi-protein action. A second project examined the novel action of small RNA molecules (80-100 bases) that appear to interact both with DNA and with the abundant HU protein. This suggests another, hitherto unknown, pathway for nucleoid organization. We have examined the complexes formed between DNA and HU in the presence of the small RNA and the images point to definite cooperative action involving small RNA. Our project with NCI on the possible chromatin de-compacting effected by HMGN5 protein overexpression was extended to provide more definitive control data with new experiments, as per publication reviewers comments. We demonstrated that the GFP co-expression of the HMGN5 did not significantly alter nuclear stiffness and that the observed stiffness change over the nucleus was actually most likely due to changes within the nucleus itself. A recently initiated collaboration with NIDCR relates to the stimulation of invadopodia generation by the extracellular matrix. Invadopodia are actin-rich cell protrusions capable of locally degrading the extracellular matrix thus facilitating cell invasion (as in cancer). Our collaborators observed a very high level of invadopodia induction in cancer cells and in primary fibroblasts when the cells were embedded in a novel high-density fibrillar collagen matrix. To test their findings they compared a number of extracellular matrix compositions and we used the AFM to measure the elastic properties of each of those matrices. The question addressed was whether it was the stiffness or the composition of the matrix that was the catalyst for invadopodia induction. Our collaborators at NEI succeeded in expressing and purifying retinoschisin (RS), a protein expressed in the retina and involved in early onset macular degeneration. The previous expression system was less robust and the protein did not fold properly. We imaged the newly expressed protein with the AFM and, for the first time, we could visualize a clear octameric ring. The stoichiometry had been deduced earlier by ultracentrifugation and our images confirmed that finding. Our collaborators then turned to cryo-EM to produce a high resolution 3-D image of the ring. We are at the same time examining the hypothesis that the ring serves as an adhesion complex linking directly plasma membranes of neighboring cells. That implies binding of RS directly to lipids, possibly glycosylated ones. To examine we image lipid vesicles of various compositions in the presence and absence of RS.